This study investigates the impact of cytosine methylation on the DNA binding specificities of human transcription factors (TFs). Using high-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX) and methylation-sensitive SELEX, the researchers analyzed the binding preferences of 542 human TFs to CpG-methylated and unmethylated DNA sequences. They found that many TFs prefer CpG-methylated sequences, particularly those belonging to the extended homeodomain family. Structural analysis revealed that homeodomain TFs recognize methylcytosine through direct hydrophobic interactions with the 5-methyl group. The study also identified a mechanism of TF binding specificity diversification, where the addition of an AT-hook domain can lead to a preference for AT-rich sequences flanking the TF's recognition motif. Additionally, the researchers observed that TF binding to methylated sites can be positively or negatively affected by methylation, and that this effect varies among different TF families. The findings highlight the widespread influence of CpG methylation on TF binding and provide insights into the mechanisms of epigenetic regulation and cellular reprogramming.This study investigates the impact of cytosine methylation on the DNA binding specificities of human transcription factors (TFs). Using high-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX) and methylation-sensitive SELEX, the researchers analyzed the binding preferences of 542 human TFs to CpG-methylated and unmethylated DNA sequences. They found that many TFs prefer CpG-methylated sequences, particularly those belonging to the extended homeodomain family. Structural analysis revealed that homeodomain TFs recognize methylcytosine through direct hydrophobic interactions with the 5-methyl group. The study also identified a mechanism of TF binding specificity diversification, where the addition of an AT-hook domain can lead to a preference for AT-rich sequences flanking the TF's recognition motif. Additionally, the researchers observed that TF binding to methylated sites can be positively or negatively affected by methylation, and that this effect varies among different TF families. The findings highlight the widespread influence of CpG methylation on TF binding and provide insights into the mechanisms of epigenetic regulation and cellular reprogramming.